The basic principles of X-ray crystallography are based on the fact that X-rays can diffract, or bend, when they encounter atoms in a crystal. This diffraction produces a pattern of spots on a detector that can be used to determine the arrangement of atoms in the crystal.
The key steps involved in X-ray crystallography are:
- Preparation of a suitable crystal: The molecule of interest is purified and crystallized to obtain a regular array of the molecules in a repeating pattern. This is a critical step as the quality of the crystal will determine the resolution of the final structure.
- X-ray diffraction data collection: The crystal is placed in a beam of X-rays, and the diffracted X-rays are collected on a detector. The crystal is rotated to collect data from different angles.
- Calculation of structure factors: The diffraction pattern obtained from the crystal is transformed into a set of numbers called structure factors, which are the amplitudes and phases of the diffracted X-rays. The phases are the most critical pieces of information required to solve the structure.
- Structure solution: The structure factors are used to solve the phases and calculate the electron density map of the crystal. This is achieved using computational methods and algorithms.
- Model building and refinement: A model of the molecule is built into the electron density map, and the model is refined iteratively until it fits the experimental data as closely as possible.
- Validation: The final structure is validated using various criteria, such as the quality of the electron density map, the fit of the model to the data, and the stereochemistry of the molecule.
X-ray crystallography is a powerful technique that has been used to determine the structures of a wide range of biological molecules, including proteins, nucleic acids, and small molecules. The technique has played a critical role in the development of new drugs and therapies and has advanced our understanding of the fundamental principles of molecular biology.